ARTICLE

ABSTRACT

Parkin and alpha-synuclein (α-syn) are two key proteins involved in the pathophysiology of Parkinson’s disease (PD). Oligomerization/aggregation and excessive secretion of α-syn contributes to PD through free radical stress, mitochondrial impairment, and synaptic dysfunction. Parkin, an E3 ubiquitin ligase, is considered to be a pleiotropic, neuroprotective protein that modulates metabolic turnover and the accumulation of α-syn. This is in addition to parkin’s role in counteracting the more distant effects of α-syn on cellular survival by altering proteasomal, autophagic, and calpain-mediated protein degradation pathways that can reduce α-syn levels. Moreover, parkin regulates mitochondrial turnover, cell survival, and immune phenomena – processes that are all known to be disturbed in PD. In addition, parkin might have an impact on the spreading and propagation of α-syn by controlling its post-translational modifications. On the other hand, recent research has shown that α-syn oligomers affect the expression, post-translational modification, and activity of parkin. This review focuses on the molecular mechanisms of cross-talk between parkin and α-syn in PD. The physical and functional interactions between α-syn and parkin, which have been incompletely characterized to-date, may present a new therapeutic avenue in PD and related synucleinopathies. The development of effective, clinically feasible modulators may offer great hopes for the therapy of PD.

Graphical ABSTRACT

INTRODUCTION

Parkinson’s disease (PD) is one of the most common age-related neurodegenerative disorders. Despite years of intense research and testing of candidate treatments, all available therapies are symptomatic. The long period of stealthy, relatively symptom-free development is shared with other neurodegenerative disorders (notably, with Alzheimer’s disease – AD), and hampers diagnosis, elucidation of disease mechanisms, and possible therapy (reviewed in Jęśko et al., 2017). The main neuropathological hallmarks of PD are 1) intraneuronal cytoplasmic inclusions, termed Lewy bodies (LB), that contain α-synuclein (α-syn) as a major component and multiple other proteins including the E3-ubiquitin ligase parkin, and 2) the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta which leads to reduced dopamine (DA) levels in the striatum – a brain region that controls locomotor activity. The majority of PD cases are characterized as spontaneous. Only about 20% of patients show a family history of the disease and, of those with a family history, only a minority follow Mendelian patterns of inheritance. α-syn and parkin are two of the genes with mutations that have been associated with typical forms of PD (Coppedè, 2012). Beyond PD, other relatively common synucleinopathies include the PD spectrum diseases PD dementia (PDD) and dementia with Lewy bodies (DLB), and the relatively more distant multiple system atrophy (MSA).

α-syn is a soluble, 140-amino acid protein with predominantly presynaptic localization, but is also found in lower quantities in other cell regions (e.g., nuclear envelope, perikaryon). α-syn is abundantly expressed in the brain, particularly in the cerebral cortex, hippocampus, substantia nigra, striatum, and olfactory bulb, and is also present outside the central nervous system (CNS) (Adamczyk et al., 2005b; Taguchi et al., 2016: Jęśko et al., 2017). The proposed physiological functions of α-syn are numerous and complex. Indeed, α-syn is implicated in synaptic signaling (i.e., neurotransmitter release, functioning of the synaptic vesicle pool), Ca2+ homeostasis, and the regulation of gene expression. Importantly, α-syn plays a significant role in dopaminergic neurotransmission, which is selectively deregulated in PD. A wealth of data indicate that loss or perturbation of physiological activity of α-syn contributes to the development and progression of PD. However, results obtained to-date focus primarily on α-syn’s gain-of-function mechanism, i.e., the acquisition of neurotoxic properties by α-syn. Misfolded α-syn oligomerizes and then forms fibrils, and is sometimes suggested to have prion-like properties (the capability of initiating aggregation of further, ‘native’ α-syn molecules). Cell-to-cell transfer of α-syn, whether leading to spread of misfolding or simply to α-syn presence in recipient cells, is a novel mode of pathology propagation, previously underestimated but potentially crucial, leading to the characteristic spatial sequence of pathological events in PD development (Jęśko et al., 2017). The gain-of-function neurotoxic alterations in α-syn may be evoked by mutations in the α-syn gene, SNCA, via post-translational modifications of the α-syn protein and/or by its oligomerization or aggregation. PD-linked dominant SNCA mutations range from missense changes that cause single amino acid substitutions to gene dosage changes (i.e., duplication or triplication; Farrer et al., 1998, 2004). Deregulated post-translational α-syn modifications, including phosphorylation, oxidation, or nitration, may also alter the metabolism and function of α-syn (Jęśko et al., 2017). Hypotheses describing potential mechanisms of α-syn toxicity include (for review, see Wong and Krainc, 2017):

Disruption of numerous vesicular pathways, including deregulation of the DA release mechanism which may lead to intracellular accumulation and cytotoxic oxidation of the neurotransmitter. In addition, disruption of autophagy, which governs the removal of dysfunctional proteins and organelles.

Damage to the endoplasmic reticulum (ER) and Golgi apparatus, leading to deregulated transport of macromolecules and induction of ER stress.

Parkin is a 52kDa, 465aa E3 ubiquitin-protein ligase with broad-spectrum neuroprotective activities and is found in LB along with α-syn. Parkin substrates include O-glycosylated α-syn, Aβ1-42, and cytoskeletal proteins (reviewed in Shimura et al., 2012). Parkin is involved in the maintenance of mitochondrial metabolism, which is particularly critical in the richly arborized DAergic neurons of the substantia nigra (Giguère et al., 2018). As a component of the ubiquitin-proteasome system (UPS), parkin plays a key role in the degradation of damaged or misfolded proteins, and in the removal of defective mitochondria via mitophagy (Geisler et al., 2010). Parkin is engaged in 1) the classical K48-G76 ubiquitination which leads to proteasomal degradation, but also in 2) monoubiquitination and 3) K63-linked polyubiquitination that targets proteins – or whole organelles – for autophagic degradation (Olzmann et al., 2007; Zheng and Hunter, 2013; Zhu et al., 2017). K63-linked polyubiquitination additionally serves distinct signaling purposes in the regulation of protein synthesis-dependent response to oxidative stress (Silva et al., 2015).

Parkin can bind to promoter DNA and behave as a transcriptional activator or repressor, thereby regulating the gene expression of p53 or presenillin-1 and -2 (da Costa et al., 2009; Duplan et al., 2013). Moreover, parkin is translocated to the nucleus in response to DNA damage. In the nucleus, parkin binds to Nijmegen breakage syndrome 1 (NBS1), a protein engaged in DNA double strand break signaling and repair, and ensures its efficient recruitment to UV-induced DNA lesions (Zhu et al., 2017). Parkin also monoubiquitinates proliferating cell nuclear antigen (PCNA), which is an essential co-factor for DNA polymerase δ that is engaged in repair-linked and general DNA replication (Zhu et al., 2017). Taken together, the resulting effects of parkin on DNA repair may underlie the observed increased risk of skin tumors in PD (Zhu et al., 2017).

Various rearrangements of the parkin gene (PARK), including exon deletion, multiplication, and point mutations, have been implicated in the pathogenesis of familial PD (Pankratz et al., 2009). Analysis of the variations of PARK as well as plasma levels of parkin protein may yield a useful diagnostic factor for PD (Geldenhuys et al., 2014). Indeed, disturbances in post-translational modifications of parkin protein, such as phosphorylation, S-nitrosylation, or oxidative damage, might be linked to the development of PD (also in the sporadic disease forms) (reviewed by Dawson and Dawson, 2014). Stimulation of parkin phosphorylation at Ser65 (by PINK1) has been shown to facilitate parkin recruitment to the mitochondria (Hertz et al., 2013). Thus, activation of PINK1 with, for e.g., kinetin triphosphate, an ATP analog (Hertz et al., 2013), might promote parkin recruitment to the impaired mitochondria.

THE POSSIBLE INVOLVEMENT OF PARKIN IN THE MODULATION OF A-SYNUCLEIN METABOLISM AND AGGREGATION

Parkin and α-syn secretion

Recent studies indicate that α-syn is secreted into the extracellular space, and secretion appears to be a precisely regulated, physiological process (Jęśko et al., 2017). However, in PD, abnormal excess release of monomeric and/or aggregated α-syn might lead to free radical stress (Wilkaniec et al., 2019) and trigger immunological response of surrounding astrocytes and microglia, thereby accelerating the neurotoxic insult (Allen Reish and Standaert, 2015). Moreover, the internalization of secreted α-syn by neurons and glia spreads the neurodegenerative pathology and involves other cell types, including cells that do not typically express significant levels of α-syn (Jęśko et al., 2017). Transport of α-syn to neighboring cells has been reported to propagate neurodegeneration (Desplats et al., 2009). α-syn is sometimes likened to the prion protein due to its ability to transfer between different types of cells, and refold from the physiological α-helix-rich conformation into an oligomerization-prone β-sheet structure, (Jęśko et al., 2017).

The role of parkin in secretion of α-syn into the extracellular space has not been studied extensively to-date. Previous research demonstrates that normal function of parkin is essential for LB formation, given that mutations in the parkin gene result in the absence of LB (Chung et al., 2001). This has led to suggestion that the formation of intracellular inclusions might be a tissue protective mechanism reducing α-syn release into extracellular space and the propagation of the pathology to neighboring cells, effectively at the expense of single cell survival (Chung et al., 2001). Despite these data, it remains unclear whether parkin’s participation in inclusion formation is actually neuroprotective or not.

Parkin and post-translational modifications of α-syn

There is much evidence that the elusive physiological functions of α-syn undergo tight regulation via covalent modifications. Moreover, many of these modifications, such as Ser129 phosphorylation, nitration, mono-/diubiquitination, and cleavage (e.g., C-terminal proteolysis), have been shown to enhance α-syn oligomerization and may therefore influence α-syn toxicity (Fig. 1).

Fig. 1.

Parkin and α-syn mutually regulate their post-translational modifications. Numerous cytotoxic actions of α-syn require post-translational modifications of its molecule, such as phosphorylation, nitration, or partial cleavage. Parkin-dependent inhibition of monoamine oxidase (MAO) may decrease oxidation and nitration of α-syn. Parkin, through increased protein phosphatase 2A (PP2A) activity and the resulting inhibition of polo-like kinase 2 (PLK2), can also reduce α-syn phosphorylation. In turn α-Syn, through its influence on dopamine metabolism and signaling pathways can lead to decreased parkin E3 ubiquitin-ligase activity.

Fig. 2.

Parkin and α-syn can affect dopamine neurotoxicity and selected pathways of programmed cell death signaling. (A) Both α-syn and parkin affect dopaminergic neurotransmission, although especially parkin’s significance is poorly characterized. Parkin can prevent cytosolic DA accumulation, partially through inhibition of the complex influence of α-syn on dopamine transport. Parkin may also reduce dopamine oxidation by inhibiting monoamine oxidase (MAO). (B) Parkin’s neuroprotective influence (largely exerted through PI3K/Akt signaling and through negative regulation of p53 expression) is disturbed in PD, underlying some of the neuronal susceptibility to death. In turn, α-syn has a more ambiguous influence on p53, and – depending on concentration – can inhibit anti-apoptotic Bcl-2 family proteins. α-syn accumulation may lead to the release of cytochrome c from mitochondria and activation of caspases, and/or to the release of AIF which can signal parthanatos, a cell death mode engaging chromatin-binding proteins normally associated with DNA repair. However, although links between parkin, α-syn, and the AIF release/parthanatos have been observed, their exact mechanism is unclear.

stimulation of the expression or activity;

reduction of expression/inhibition of activity;

ambiguous or changing interactions (positive or inhibitory influence depending on the concentration of the protein in question, cellular metabolic status, or biological model used).

Proteolytic cleavage of α-syn may not only lead to its degradation, but also to its pathological aggregation. Indeed, previous research shows that C-terminally truncated α-syn has a stronger tendency towards aggregation and is observed in pathologically altered neurites (intriguingly, also in AD without conventional LB) (Lewis et al., 2010).

Importantly, post-translational modifications of parkin may change its activity and role in the modulation of α-syn metabolism (Fig. 1). Indeed, parkin undergoes an α-syn-induced S-nitrosylation (Wilkaniec et al., 2019), which can lead to parkin autoubiquitination and degradation in PC12 cells (Wilkaniec et al., 2019). Other research has shown that covalent binding of DA reduces parkin solubility and inhibits its activity as an E3 ubiquitin ligase (LaVoie et al., 2005). Numerous kinases may be involved in the regulation of parkin phosphorylation on serines 101, 127, 131, 378 and Tyr 143. One potential kinase is cdk 5, which regulates parkin solubility together with casein kinase-1 (Chakraborty et al., 2017). Previous research in PC12 cells shows activation of cdk5 in response to α-syn-induced Ca2+ rise sensed by calpain. In addition, inhibition of cdk5 reduces α-syn cytotoxicity (Czapski et al., 2013). It remains to be seen if parkin aggregation occurs in response to α-syn-evoked activation of cdk5 in vivo, and if the potential loss of parkin activity contributes to α-syn neurotoxicity in the Parkinsonian brain.

Parkin and α-syn degradation

The extensive network of redundant pathways that are capable of degrading α-syn highlights the importance of continuous control of α-syn concentration. Strikingly, parkin has been found to modulate the degradation of α-syn by multiple mechanisms.

α-syn is a substrate for parkin-mediated ubiquitination, and the UPS appears to be a major pathway of α-syn degradation (Jęśko et al., 2017). Whereas loss of parkin activity leads to accumulation of non-ubiquitinated forms of α-syn (Shimura et al., 2001), compounds that augment proteasomal α-syn degradation have beneficial neuroprotective effects and are considered to have a potential therapeutic application in PD (Jęśko et al., 2017; Li et al., 2018). However, accumulation and aggregation of α-syn can negatively affect UPS function, potentially leading to a runaway failure (Snyder et al., 2003). The mechanism and significance of α-syn level control by UPS still raise questions. For example, not all α-syn cleaved by proteasomes is ubiquitinated. In addition, the aforementioned incomplete cleavage of α-syn by a pathologically overloaded proteasome system may lead to the generation of α-syn fragments that are prone to aggregation (Liu et al., 2005).

α-syn is also degraded via the autophagic pathway. Indeed, PD-linked alterations in parkin lead to the accumulation of unprocessed α-syn in autophagosomes (Lonskaya et al., 2013). Similar to UPS, the potential of autophagy as a backup degradation pathway for α-syn is limited by its sensitivity to inhibition by accumulated or aggregated α-syn (Jęśko et al., 2017).

Parkin-activated cysteine protease calpain is also involved in degradation of α-syn (Kim et al., 2003). However, the ability of this cleavage mechanism to limit neurotoxic α-syn accumulation and/or aggregation is unclear (Beyer and Ariza, 2013).

Table I.

α-syn has been shown to affect various aspects of DAergic signaling (Fig. 2A), including DA biosynthesis, release, and re-uptake. Parkin has also been shown to affect multiple α-syn-modulated steps of DA metabolism and transport, although the results are somewhat contradictory. For example, research shows that α-syn binds to and inhibits tyrosine hydroxylase (TH), the rate-limiting DA biosynthesis enzyme (Perez et al., 2002). In pathological conditions, α-syn alters DA synaptic homeostasis, including dopamine transporter (DAT) modification (Adamczyk et al., 2006, Moszczynska et al., 2007) and impairment of DA packaging into synaptic vesicles, which leads to cytosolic DA accumulation, DA oxidation, and cellular stress (Guo et al., 2008). Parkin appears to have an opposite influence on DA, as disruption of parkin function leads to cytosolic DA accumulation at the cost of packaging into vesicles (Itier et al., 2003). The role of parkin in DAT regulation is less characterized, although it is known that parkin can bind to DAT and prevent DAT’s interaction with α-syn (Moszczynska et al., 2007). Consistent with this, reduced DAT levels have been detected in parkin-deficient mice (Itier et al., 2003) (see Fig. 2A and Table I).

Parkin and the α-syn-induced oxidative and nitrosative stress

Although α-syn has been widely associated with the induction of oxidative stress, some results suggest that its physiological role might instead be linked to antioxidative defense (Deas et al., 2016; Jęśko et al., 2017). Interestingly, aggregation of α-syn appears to generate and be driven by free radicals. Indeed, previous research shows that hydroxyl radical (•OH) is produced in the course of in vitro α-syn aggregation in the presence of iron (II) (Turnbull et al., 2001), and that iron-induced α-syn aggregation can be blocked by the reactive oxygen species (ROS) scavenger vitamin E (Li et al., 2011). Reactive nitrogen species (RNS) and nitrosative stress have also been shown to contribute to Parkinsonian pathology (Gatto et al., 2000). Indeed, extracellular α-syn can open N-methyl-D-aspartate receptor (NMDAR) ion channels, which leads to Ca2+ influx and activation of neuronal nitric oxide synthase (nNOS) in the rat brain (Adamczyk et al., 2009). Nitric oxide (NO) can react with superoxide to form the highly harmful peroxynitrite (ONOO-), which damages proteins, lipids, and DNA. α-syn also contributes to the oxidative imbalance by depleting cellular glutathione levels and can also induce aggregation of superoxide dismutase protein. However, results on these effects are inconsistent (Deas et al., 2016; Koo et al., 2013; Koch et al., 2016). Moreover, oxidized glutathione produced in the course of ROS detoxication can itself contribute to α-syn aggregation (Paik et al., 2003). In turn, oxidative or nitrosative stress may influence intracellular α-syn localization, function, and release into the extracellular space (Jang et al., 2010; Sangchot et al., 2002). Oxidative or nitrosative stress may also mediate cytotoxic outcomes of DA metabolism deregulation by α-syn (Riobó et al., 2002).

It has been suggested that parkin plays a neuroprotective role against oxidative stress. Indeed, parkin-null mice have been shown to display increased vulnerability to oxidative stress (Palacino et al., 2004), and parkin can suppress the expression and activity of MAO-A and -B, which generate ROS during DA oxidation (Itier et al., 2003; Jiang et al., 2006) (see Fig. 2A and Table I). However, parkin protein itself has been shown to be extremely sensitive to biochemical stressors. Indeed, oxidative conditions and DA toxicity can inhibit parkin, and induce its assembly into multimers that may be degraded via macroautophagy or the roteasome (Wang et al., 2005).

The protective role of parkin against α-syn-evoked microtubule destabilization

Microtubules are involved in the axonal transport of proteins and organelles. Microtubule disruption has been shown to inhibit neuronal activity, and may also lead to axonal accumulation of cargo normally transported along the cytoskeleton, such as aggregated proteins (thereby boosting α-syn aggregation) or damaged mitochondria-containing autophagosomes (thus further deepening auto- or mitophagic dysfunction and mitochondrial deregulation) (Esteves et al., 2014).

Mitochondrial abnormalities observed in PD may lead to a lack of energy, which detrimentally affects the functioning of the microtubular cytoskeleton, for e.g., the molecular motors that transport the cargo (Esteves et al., 2014).

The role of α-syn in microtubule depolymerization and polymerization in nigral DAergic neurons has been studied by several groups. These studies have shown that α-syn binds to and is necessary for the activity of glycogen synthase kinase 3β (GSK-3β), a serine/threonine kinase that phosphorylates microtubule-associated protein (MAP) Tau (Duka et al., 2009; Khandelwal et al., 2010). α-syn-dependent Tau hyperphosphorylation by GSK-3β results in destabilization of the cytoskeleton and impairment of cellular survival (Duka et al., 2009; Khandelwal et al., 2010; Gąssowska et al., 2014).

Potential anti-inflammatory role of parkin

Inflammation has been shown to play an important role in the progression of PD. Indeed, α-syn has been shown to act as an inflammatory stimulator that modulates microglial expression of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and inducible NOS (iNOS) (Khandelwal et al., 2010; Su et al., 2008). Nitrosative and oxidative stress has been observed in cells overexpressing or treated with α-syn (Wilkaniec et al., 2019). α-syn may exert its effects in part through the NF-κB pathway, which is crucially engaged in inflammatory signaling (Yuan et al., 2008).

It has been suggested that parkin plays a protective role against inflammatory responses evoked by α-syn (Table I). Indeed, research shows enhanced inflammatory damage in parkin knock-out mice (Frank-Cannon et al., 2008), whereas virally expressed parkin significantly reversed the effect of α-syn on glial number and morphology, and reduced the up-regulation of TNF-α and iNOS (Khandelwal et al., 2010). It has been proposed that the neuroprotective effects of parkin depend on the NF-κB pathway (Henn et al., 2007). However, α-syn-induced stress can lead to S-nitrosylation and degradation of parkin (Wilkaniec et al., 2019).

Parkin and α-syn in the regulation of apoptosis, autophagy, and parthanatos

The role of both α-syn and parkin on the PI3K-Akt/PKB pathway has been incompletely characterized to-date. The PI3K-Akt/PKB pathway is a highly interconnected modulator of cellular metabolism and survival and death through, for e.g., NF-κB, GSK-3β, Forkhead family transcription factors, Bad, or caspase-9. Parkin supports pro-survival PI3K-Akt signaling (Fallon et al., 2006) whereas α-syn has a more complex role. Nanomolar α-syn activates PI3K-Akt whereas micromolar α-syn has been shown to exert opposite effects, including reduction of Bcl-xL expression, up-regulation of Bax, and inducing the release of cytochrome c from mitochondria (Seo et al., 2002). Based on these data, it is highly probable that the fine-tuned activities of α-syn in this context may be successfully modified by the documented interactions with parkin.

Autophagy as a cell death mechanism also appears to be involved in PD (Venderova and Park, 2012). Indeed, α-syn has been shown to be involved in autophagosome regulation by negatively affecting early stages of autophagosome formation. These data suggest that a loss-of-function mechanism might be involved in PD pathogenesis (Winslow and Rubinsztein, 2011).

Parthanatos is a relatively recently characterized mode of cell death that was named after PAR, poly(ADP-ribose) and Thanatos. Parthanatos involves proteins that are typically engaged in DNA repair, including poly(ADP-ribose) polymerase (PARP1) or 8-oxoG glycosylase1 (OGG1), These proteins lead to PAR-mediated signaling from the nucleus to the mitochondria, triggering release of apoptosis-inducing factor (AIF), chromatin condensation and large scale fragmentation, and ultimately, cell death. Parthanatos seems to be significantly involved in PD and MPTP-induced models of PD (Lee et al., 2014). α-syn is known to interact with AIF signaling (Zhu et al., 2011) and has been shown to interact bi-directionally with PARP-1 signaling, although the topic requires further research (Adamczyk et al., 2005a; Adamczyk and Kaźmierczak, 2009; Chiba-Falek et al., 2005; Outeiro et al., 2007).

Defining roles of parkin and α-syn in mitochondrial remodeling and dynamics

To uphold the pool of bioenergetically competent mitochondria, the ‘life cycle’ of mitochondria must pass through the stages of biogenesis, remodeling via repeated cycles of fusion/fission, and mitophagy (see Fig. 3). Although the mechanistic details of the neuroprotective mechanism behind fusion/fission remain obscure, it has been hypothesized that fission could improve mitochondrial quality through asymmetric segregation of faulty proteins into one daughter organelle. Fusion, in contrast might allow for complementation of the best components from the two original mitochondria (Scarffe et al., 2014).

Fig. 3.

Involvement of parkin and α-syn in the mitochondrial ‘life cycle’. Mitochondrial ‘life cycle’ involves their biogenesis, remodeling through repeated rounds of fusion/fission, and mitophagy. It is suggested that parkin causes ubiquitination and degradation of the PARIS protein, removing its negative influence on the levels of PGC-1α; thus, parkin leads to elevated PGC-1α, increased replication of mtDNA, and intensified mitochondrial biogenesis. Moreover, depending on the state of cellular metabolism parkin may upregulate mitochondrial motility and mitophagy through interaction with Miro-1 and autophagolysosomal proteins, respectively. α-syn – evoked oxidative/nitrosative stress downregulates de novo mitochondrial biogenesis. α-syn also supports mitochondrial fission, inhibits fusion, and can influence mitophagy leading to accumulation of damaged mitochondria and cell death.

stimulation of the expression or activity;

reduction of expression/inhibition of activity;

ambiguous or changing influence.

Overexpression of wildtype (wt) or mutant α-syn has been shown to promote neuronal cell death via mitochondrial dysfunction and fragmentation (Kamp et al., 2010; Zhu et al., 2011). α-syn is an inhibitor of membrane fusion and knockdown of α-syn has been shown to promote mitochondrial elongation (Norris et al., 2015). Interestingly, mitochondrial fragmentation evoked by α-syn overexpression can be rescued by co-expression of parkin, PINK1, or DJ-1, suggesting that α-syn and parkin may take part in the same pathway (as described below) (Kamp et al., 2010). Recently, it was also indicated that α-syn accumulation or the α-syn A53T mutation leads to inhibition of mitochondrial motility (Xie and Chung, 2012).

Parkin also appears to be an essential component of the quality control s ystem in the mitochondria (Venderova and Park, 2012), and inactivation of parkin leads to reduced striatal neuron respiration and oxidative d amage (Damiano et al., 2014; Palacino et al., 2004). Parkin has been shown to regulate multiple aspects of the mitochondrial life cycle, from biogenesis to mitophagy (Lim et al., 2012) (see also Table I). These regulatory effects are described below:

Parkin regulates mitochondrial fusion, fission, and mitophagy in a manner that depends on cellular condition. In physiological conditions, parkin can inhibit fusion-promoting Mfn family proteins and the fission-related Drp1. Under moderate mitochondrial stress, parkin may play a fusion-promoting role that allows the repair of damaged organelles while counteracting fission. Of note, fission occurs intensely in parkin-deficient cells in response to mitochondrial toxins (Norris et al., 2015). In contrast, heavily damaged mitochondria trigger its mitophagy-promoting ac tivity (Norris et al., 2015).

Parkin may also play an essential role in intracellular transport of mitochondria through regulation of proteins of the axonal and dendritic trafficking machinery, including Miro1 (McLelland et al., 2018).

Impaired clearance of damaged mitochondria may be one of the triggers causing demise of DAergic neurons in PD brain. Post-mitotic neurons have fewer opportunities to remove defective mitochondria than cycling cells, and at the same time, post-mitotic neurons have extreme energy demands, making them more vulnerable to disturbances. Previous studies confirm accumulation of defective mitochondria PD patients (de Vries and Przedborski, 2013).

Mitophagy is a subtype of lysosome-mediated autophagy that is required to clear damaged mitochondria. Research shows that many genes linked to PD are engaged in the regulation of mito phagy (Ryan et al., 2015). Disturbed or delayed mitophagy in the presence of accumulated α-syn may be an essential mediator of α-syn toxicity and thus represent a pathological mechanism leading to PD (Shaltouki et al., 2018). Other research demonstrates enhanced mitophagy in a cell line that overexpresses α-syn (Martinez et al., 2018); in particular, in midbrain DAergic neurons transfected with A53T α-syn (which, in that case, leads to cell death) (Choubey et al., 2011). Mitophagy was also found to be stimulated by nonfibrillar, phosphorylated α-syn aggregates found in the neurons of PD patient brains (Grassi et al., 2018). The role of α-syn in autophagy of mitochondria is therefore unclear, and its disease-associated molecular alterations appear to autophagy profoundly.

Interestingly, under exposure to the mitochondrial toxin, parkin, together with E2 conjugating enzyme Ubc13 has been shown to stimulate K63-linked α-syn ubiquitination. The observed effect of parkin on mitochondrial turnover can be phenocopied by α-syn-targeting siRNA (Norris et al., 2015). Parkin itself has been shown to play an essential role in removing damaged mitochondria from the cell via mitophagy, including local liquidation of mitochondria in the axon, which has direct relevance for neuronal activity (Ashrafi and Schwarz, 2015; Chan and Chan, 2011). Mitochondria that accumulate misfolded proteins in the matrix can trigger the translocation of parkin, which activates subsequent removal (Jin and Youle, 2013) (Fig. 3).

THE RECIPROCAL INFLUENCE OF A-SYN ON PARKIN, AND A-SYN-PARKIN CO-REGULATION

α-syn appears to modify parkin’s ubiquitination activity and function by binding to parkin and shifts parkin’s activity towards K63-linked ubiquitination. α-syn’s modification of parkin stimulates mitochondrial fusion, and this interaction occurs in response to mild mitochondrial stress (Norris et al., 2015). α-syn can also modify the phosphorylation state of parkin, given that overexpression of A53T α-syn has been shown to leads to parkin modification by p38MAPK. Modification of parkin, in turn, reduces its cytoprotective influence on mitochondrial turnover (Chen et al., 2018).

A recent study by Liu et al. (2019) described another mechanism linking deregulation of parkin with α-syn in PD. In this work, Liu et al. (2019) found an upregulation of ubiquitin-specific protease USP13 in Parkinsonian brains. USP13 removes ubiquitin from proteins, and thus, USP13 is able to reduce α-syn-induced parkin ubiquitination. Likewise, USP13 de-ubiquitinated α-syn, and a USP13 knockdown has been shown to facilitate the ubiquitination and removal of α-syn. However, the observed effect on α-syn ubiquitination was shown to be independent of parkin (Liu et al., 2019).

CONCLUSIONS

Parkin is extensively engaged in protein degradation pathways, and as such may offer significant therapeutic potential. α-syn levels may be controlled by parkin-dependent proteasomal degradation. Parkin also appears to modulate autophagic degradation of α-syn, as well as, control calpain, an α-syn-degrading enzyme. All of these pathways are shown to be disturbed in PD, at least partially due to parkin’s sensitivity to α-syn-induced stress. In-depth understanding of this mechanism is a necessary aspect of PD characterization and may lead to identification of new therapeutic opportunities. Parkin and α-syn mutually regulate their own post-translational modifications. This relatively easy to manipulate mechanism influences the neuroprotective activity of parkin and the tendency of α-syn to aggregate, thus offering important insights into pathophysiological mechanisms leading to PD and potential therapeutic value. Importantly, α-syn also alters parkin levels and activity (Wilkaniec et al., 2019), potentially creating a cycle of self-fueling pathology.

The multiple neuroprotective functions of parkin raise hopes that, in addition to modulating α-syn behavior, parkin may also alter the downstream effects of α-syn neurotoxicity. Effects on α-syn neurotoxicity may allow for a more established and/or easier to implement avenue for therapeutic intervention. A promising aspect of parkin’s role in the cell is linked to its engagement in mitochondrial turnover and/or quality maintenance, and in cytoskeletal functions. These functions are particularly important in neuronal cells, which are extremely dependent on efficient oxidative metabolism, and efficient maintenance of intracellular transport. In line with this, mitochondrial metabolism is known to be compromised in neurodegenerative disorders. Moreover, parkin may influence multiple steps of DA turnover, preventing its intracellular accumulation, oxidation-mediated toxicity, and moderating other mechanisms of cellular stress. Parkin can also modulate cell survival signaling and inhibit apoptotic and autophagic cell death, and parthanatos. Neuronal death is a relatively distant effect of the partially obscure processes that lead to the development of PD. However, as only symptomatic treatments are currently available for neurodegenerative disorders, interventions that are capable of preserving neuronal populations in affected brain regions would be of extreme value in the struggle against PD.

Recent progress in the understanding of parkin and α-syn dysfunction provide hope for the discovery of new therapeutic approaches for synucleinopathies. Research addressing practical aspects of the restoration of parkin activity primarily concentrate on:

FIGURES & TABLES

Fig. 1.

Parkin and α-syn mutually regulate their post-translational modifications. Numerous cytotoxic actions of α-syn require post-translational modifications of its molecule, such as phosphorylation, nitration, or partial cleavage. Parkin-dependent inhibition of monoamine oxidase (MAO) may decrease oxidation and nitration of α-syn. Parkin, through increased protein phosphatase 2A (PP2A) activity and the resulting inhibition of polo-like kinase 2 (PLK2), can also reduce α-syn phosphorylation. In turn α-Syn, through its influence on dopamine metabolism and signaling pathways can lead to decreased parkin E3 ubiquitin-ligase activity.

Parkin and α-syn can affect dopamine neurotoxicity and selected pathways of programmed cell death signaling. (A) Both α-syn and parkin affect dopaminergic neurotransmission, although especially parkin’s significance is poorly characterized. Parkin can prevent cytosolic DA accumulation, partially through inhibition of the complex influence of α-syn on dopamine transport. Parkin may also reduce dopamine oxidation by inhibiting monoamine oxidase (MAO). (B) Parkin’s neuroprotective influence (largely exerted through PI3K/Akt signaling and through negative regulation of p53 expression) is disturbed in PD, underlying some of the neuronal susceptibility to death. In turn, α-syn has a more ambiguous influence on p53, and – depending on concentration – can inhibit anti-apoptotic Bcl-2 family proteins. α-syn accumulation may lead to the release of cytochrome c from mitochondria and activation of caspases, and/or to the release of AIF which can signal parthanatos, a cell death mode engaging chromatin-binding proteins normally associated with DNA repair. However, although links between parkin, α-syn, and the AIF release/parthanatos have been observed, their exact mechanism is unclear.

stimulation of the expression or activity;

reduction of expression/inhibition of activity;

ambiguous or changing interactions (positive or inhibitory influence depending on the concentration of the protein in question, cellular metabolic status, or biological model used).

Involvement of parkin and α-syn in the mitochondrial ‘life cycle’. Mitochondrial ‘life cycle’ involves their biogenesis, remodeling through repeated rounds of fusion/fission, and mitophagy. It is suggested that parkin causes ubiquitination and degradation of the PARIS protein, removing its negative influence on the levels of PGC-1α; thus, parkin leads to elevated PGC-1α, increased replication of mtDNA, and intensified mitochondrial biogenesis. Moreover, depending on the state of cellular metabolism parkin may upregulate mitochondrial motility and mitophagy through interaction with Miro-1 and autophagolysosomal proteins, respectively. α-syn – evoked oxidative/nitrosative stress downregulates de novo mitochondrial biogenesis. α-syn also supports mitochondrial fission, inhibits fusion, and can influence mitophagy leading to accumulation of damaged mitochondria and cell death.

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